The FDA approved gold standard potency assay for influenza hemagglutinin protein-based vaccines is single radial immunodiffusion (SRID). SRID is a time and labor intensive assay, often requiring 2-3 days to complete and a minimum of 6 hours hands on time by well-trained analysts. In the SRID assay, viral antigen is applied to an agarose gel matrix that contains specific antibodies to the antigen being measured. The applied antigen diffuses radially, and interaction between antigen and antibody produces a zone of precipitation. The size of this precipitation zone is directly proportional to the amount of antigen applied. The assay is also separately performed a number of times on dilutions of a reference standard, and the resulting calibration curve is then used to then determine the amount of antigen present in the unknown. Although used as the current gold standard, it is well-known that the SRID assay for influenza potency determination is fraught with uncertainty [Minor, P. D. “Vaccines against seasonal and pandemic influenza and the implications of changes in substrates for virus production”, Clinical Infectious Diseases, 2010, 50, 560-565. and references therein].
While the reference standards are provided at no cost by the Center for Biologics Evaluation and Research (CBER), additional materials must be purchased and prepared by the entity performing the testing. Based on a conservative operating cost of $100 per hour for a small protein manufacturer, the minimal cost per SRID assay is $600. At the research phase, a small-scale influenza vaccine producer typically conducts 20-25 SRID assays per week during the development and testing phase. Thus, the cost of SRID even for small scale R&D can be on the order of $60,000 per month. Perhaps even more important is the development and production delays imposed by the slow turn-around time for the method. Often vaccine producers must wait days for results prior to moving forward in the process, decreasing throughput and efficiency.
In their Fiscal Year 2010 CBER Annual Report, CBER summarized a 3-day workshop held to evaluate lessons learned from potency testing of the pandemic 2009 H1N1 vaccines. The suitability of current methods (SRID) was questioned, and a recommendation was made for improvements to the SRID assay. In addition, a recommendation was made to continue to explore alternative methods and technologies for potential future application. Also, in a talk presented at the USP 2nd Bioassay Workshop (Aug. 13, 2009), Dr. Armen Donabedian of Health and Human Services and BARDA called for a wide range of efforts to “improve or replace the SRID assay [to] facilitate seasonal and pandemic influenza preparedness.”
US Patent Application Publication No. 2010/0041022, U.S. patent application Ser. No. 12/587,136, filed Oct. 2, 2009, titled Novel Assay for the Separation and Quantification of Hemagglutinin Antigens, which is incorporated herein by reference as if set out in full, describes a high performance liquid chromatography (HPLC) based method for quantification of influenza HA proteins as an alternative to SRID. HPLC methods can work reasonably well but have not been widely adopted, likely, due to the typical drift, instrument maintenance required, poor ease-of-use, and calibration problems associated with HPLC.
US Patent Application Publication Number 2011/0201039 describes a combined liquid chromatography mass spectrometry (LCMS) method for the quantification of viral proteins, targeted towards the quantification of influenza HA protein for use in vaccine production. LCMS in general tends to be too complex for routine use by minimally trained personnel and suffers from many of the same maintenance, ease of use, and calibration issues previously mentioned.
US Patent Application Publication No. 2011/0070574, U.S. patent application Ser. No. 12/994,189, filed Nov. 23, 2010, titled Method for Virus Detection, which is incorporated herein by reference as if set out in full, describes a sensor-based method for determining the concentration of virus or viral antigen based on surface plasmon resonance detection. This method offers improved sensitivity relative to SRID, but is too expensive for routine use by all but the largest vaccine producers and requires large, complex instrumentation.
For steps in the vaccine development process where rapid protein quantification is needed, many vaccine producers rely heavily on enzyme linked immunosorbent assays (ELISA) as an alternative to SRID. In a traditional “sandwich” ELISA assay, an antibody specific to a particular antigen is immobilized on a solid support (such as the bottom of a microtiter plate). An unknown amount of antigen is then added, and binds to the surface-immobilized antibody. After the antigen is immobilized, an enzyme-linked detection antibody is then added and forms a complex with the antigen. Subsequent addition of an appropriate substrate for the enzyme ultimately results in the formation of a detectable signal. In general, ELISA assays are more rapid than SRID (hours versus days).
Although more rapid than SRID, the hands-on time required for sample preparation and analysis for ELISA assays is still costly. A research unit within a large vaccine company may conduct upwards of ˜50 ELISA assays per week. Each assay requires 7 serial dilutions per sample and 2 replicates per dilution for both a standard and an unknown sample. Thus, each sample requires 28 preparations. The analysis is typically performed over 2 days; plates are prepared on day one, with 4-6 hours of incubation/analysis on the second day. The estimated costs for materials and labor for each protein sample quantified by ELISA is $300-$400 USD, such that the cost for a lab that conducts 50 ELISAs per week is $70,000 USD per month or $840,000 USD per year. During production at a medium scale facility, the costs associated with materials, labor, and time delays are estimated to exceed several million USD per year.
Other technical disadvantages of ELISA methods include the fact that the signal is generated by an enzymatic reaction as a function of time, requiring critical timing of the reaction and inclusion of a standard with every batch of samples. Also, the enzymatic reaction must be stopped after a specified period time using a “stopping agent”, and quantification must be conducted within 2 hours of the addition of the stopping agent.
After a vaccine is developed, its efficacy must be evaluated in clinical trials whereby the immune response is evaluated following vaccination. The current gold standard assay for the evaluation of post-vaccination efficacy of a number of viral vaccines, including vaccines for influenza viruses, is the hemagglutinin inhibition (HI) assay which involves semi-quantification of antibodies within patient serum following vaccination. The HI assay is based on the underlying hemagglutination assay (‘HA assay’) for semi-quantification of hemaglutinin (HA) protein concentration. The HA surface protein on influenza virus particles are known to bind to red blood cells and hemagglutinate (forming an extended lattice-type structure). To perform the HA assay, a series of dilutions of an influenza-containing sample are prepared, and each dilution is mixed with a known amount of red blood cells, (typically in a microtiter plate). Red blood cells that are not bound in the lattice structure due to the presence of influenza virus precipitate to the bottom of the well, whereas the red blood cells that are bound to influenza particles form a lattice that coats the well. The HI assay used for vaccine efficacy evaluation takes advantage of the fact that antibodies to influenza virus that should be present after vaccination will inhibit hemagglutination due to the binding of the antibodies to the HA surface proteins. Serum obtained from a vaccinated individual is collected, and serial dilutions of the serum are added to wells each containing a known amount of red blood cells and antigen (influenza virus). The HI titer of the serum sample is determined as the highest dilution that prevents hemagglutination. In the absence of appropriate anti-HA antibodies (in an unvaccinated individual, for example), hemagglutination would be observed.
There are several practical limitations to traditional HA and HI assays. The 2002 World Health Organization Manual on Animal Influenza Diagnosis and Surveillance outlines the importance of preparing fresh antigen solutions, standardization of the protocol for red blood cell suspension, strict adherence to proper incubation times, and prompt visual “reading” of the plates after the assay is completed. In addition, non-antibody inhibitors can also exist that inhibit hemagglutination and can cause incorrect interpretation. Moreover, the HA and HI assays is limited by the subjective nature of the visual readout. Because the detection relies on interpretation of the hemagglutination pattern present in the wells, abnormal hemagglutination patterns can complicate the interpretation. In addition, a two-fold difference in HI titer is generally considered within the variability of the test and not considered significant.
In view of the foregoing, there is a clear and considerable need in the art to overcome the time, cost, and complexity limitations associated with existing protein and antibody quantification methods including SRID, ELISA, HI assay methods in the fields of vaccine-related protein quantification, potency determination, and efficacy evaluation. The technology of the present application addresses these and other limitations in the current state of the art.